Research ArticleDevelopmental Biology

Wdpcp promotes epicardial EMT and epicardium-derived cell migration to facilitate coronary artery remodeling

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Science Signaling  27 Feb 2018:
Vol. 11, Issue 519, eaah5770
DOI: 10.1126/scisignal.aah5770
  • Fig. 1 Coronary vascular defects in E18.5 Wdpcpm/m;SM22αLacZ/+ embryos.

    (A to D) Coronary vessels with smooth muscle cells were visualized by X-gal staining in SM22αLacZ/+ hearts (A) and SM22αLacZ/+;Wdpcpm/m hearts (B to D). The blue arrows indicate the left coronary arteries (LCAs), which has reduced blue staining and few branches in (B) and (C). Red arrows point to the disrupted continuity of blue staining in the LCA. Yellow arrows indicate additional short coronary arteries originating from arterial trunk. Blue and yellow arrows in (D) indicate two coronary arteries of similar size. Black arrowheads indicate left coronary veins (LCVs). (E and F) Quantification of smooth muscle coverage in coronary arteries and veins (E) and LCA branch number (F). Ctrl, SM22αLacZ/+; Mutant, Wdpcpm/m;SM22αLacZ/+. (G and H) Transparentized hearts for visualization of the septal artery (SA). Data are presented as means ± SD (n = 3 embryos per genotype) and two-tailed Student’s t test on log-transformed data. **P < 0.01. Scale bar, 500 μm.

  • Fig. 2 The subepicardial coronary plexus expands faster in Wdpcp mutants.

    (A to H) Whole-mount PECAM immunostaining. (A to D) Embryonic day 13.5 (E13.5) heart. Arrowhead indicates the initial coronary plexus expanding from the dorsal side. (E to H) E14.5 heart. Arrow indicates area devoid of coronary plexus. (A, C, E, and G) Ventral view. (B, D, F, and H) Dorsal view. Dashed line in (B), (C), (E), and (G) indicates the front of the expanding subepicardial plexus. Scale bars, 200 μm. (I and J) Quantification of vascular coverage on the dorsal side of the ventricle. (I) E13.5 heart. (J) E14.5 heart. Data are presented as means ± SD (n = 3 embryos per genotype) and two-tailed Student’s t test on log-transformed data. *P < 0.05. WT, wild type; Mutant, Wdpcpm/m. (K) Knockdown efficiency of WDPCP short hairpin RNA–1 (shRNA-1) and shRNA-2 in human umbilical vein endothelial cells (HUVECs). (L) Quantification of cell migration through Transwell membranes. Data are presented as means ± SD (n = 3 biological replicates for each group) and two-tailed Student’s t test on log-transformed data. *P < 0.05 and ***P < 0.005. (M to R) Representative images for the Transwell migration assay. Scale bar, 200 μm. (M and P) HUVECs transfected with scrambled RNA, treated or not with SAG. (N and Q) HUVECs transfected with WDPCP shRNA-1, treated or not with SAG. (O and R) HUVECs transfected with WDPCP shRNA-2, treated or not with SAG.

  • Fig. 3 Decreased EPDC numbers and compromised EMT in Wdpcpm/m hearts.

    Epicardium-derived cells (EPDCs) express enhanced green fluorescent protein (EGFP) after CreERT2-mediated recombination in Rosa26mTmG locus upon tamoxifen injection at E10.5. (A and B) Representative fluorescence-activated cell sorting analysis of GFP+ cells from E14.5 Wt1CreERT2/+;Rosa26mTmG/+ and Wdpcpm/m;Wt1CreERT2/+;Rosa26mTmG/+ ventricles. SSC, side scatter. (C) Quantification of GFP+ cells. Data are presented as means ± SD (n = 3 embryos per genotype) and two-tailed Student’s t test on log-transformed data. **P < 0.01. (D and E) Coronary cryosections of E15.5 Wt1CreERT2/+;Rosa26mTmG/+ and Wdpcpm/m;Wt1CreERT2/+;Rosa26mTmG/+ hearts. White arrows indicate regions lacking EPDCs. Insets are magnified boxed areas showing EPDCs. RV, right ventricle; LV, left ventricle; IVS, intraventricular septum. Scale bar, 200 μm. (F) Percentage of ventricular myocardium covered by GFP+ cells. Data are presented as means ± SD (n = 3 embryos per genotype) and two-tailed Student’s t test on log-transformed data. *P < 0.05. (G) Quantitative polymerase chain reaction analysis of epithelial-mesenchymal transition (EMT), mesenchymal and epithelial markers. Data are presented as means ± SD (n = 5 embryos per genotype for Snail2 and Twist1 and n = 3 embryos per genotype for the other markers) and two-tailed Student’s t test on log-transformed data. *P < 0.05 and ***P < 0.005. (H and I) Representative images of ZO-1 immunofluorescence staining on E13.5 embryos (n = 2 embryos per genotype). White arrows point out similar ZO-1 immunostaining in epicardial cells. White arrowheads point out decreased ZO-1 immunostaining in cardiomyocytes. Yellow arrows point out different epicardial cell shapes between WT and mutant hearts. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 4 Compromised invasion by Wdpcp mutant-derived EPDCs.

    E11.5 epicardial cells were explanted on a three-dimensional collagen gel matrix, and outgrowths were stained with Alexa Fluor 488–conjugated phalloidin and DAPI (blue fluorescence) to enable visualization of epicardial cell invasion by confocal microscopy. (A and B) x/y plane of representative epicardial outgrowths from WT explants (A) and Wdpcp mutant explants (B). (C and D) Confocal imaging of epicardial outgrowth along the z axis. (E) Depth measurement of epicardial cell invasion along the z axis. Cell infiltration depth along the z axis was determined by the distance between the top and bottom planes within which there were 30 cells. Data are presented as means ± SD (n = 5 embryos per genotype) and two-tailed Student’s t test on log-transformed data. ***P < 0.005. Scale bar, 100 μm.

  • Fig. 5 Epicardium-specific deletion of Wdpcp leads to coronary artery defects.

    (A to D) Left side view of E18.5 hearts after X-gal staining in Wdpcpm/+;SM22αLacZ/+ heart (A) and Wdpcpm/flox;WT1CreERT2/+;SM22αLacZ/+ mutant hearts with tamoxifen injected at E10.5 (B to D). Black arrows indicate the LCA. Black arrowheads indicate the LCV. Yellow arrow indicates an additional coronary artery originating from the aorta. Red arrow indicates discontinuity of blue staining in LCA. Scale bar, 500 μm. (E to L) Representative coronal sections of X-gal–stained Wdpcpm/flox;WT1CreERT2/+;SM22αLacZ/+ hearts (n = 2 mice). (E to H) Serial sections from dorsal to ventral side show patent LCA originating from the aorta (AO). (I to L) Serial sections from ventral to dorsal side show patent right coronary artery originating from the aorta. PA, pulmonary trunk. Scale bar, 200 μm.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/519/eaah5770/DC1

    Fig. S1. Expression of Wdpcp in embryonic heart.

    Fig. S2. Ciliary defects but normal transcriptional response to Shh signaling in Wdpcp mutant hearts.

    Fig. S3. Normal formation of the intramyocardial endothelial plexus.

    Fig. S4. Normal egression of coronary endothelial cells from the sinus venosus.

    Fig. S5. Proliferative and apoptotic status of coronary endothelial cells in E13.5 hearts.

    Fig. S6. Normal coronary vessel formation in E18.5 embryos with myocardium-specific deletion of Wdpcp.

    Fig. S7. Wdpcp knockdown recapitulates the gel infiltration defect of Wdpcp mutant-derived EPDCs.

    Table S1. List of qPCR primers used in the study.

  • Supplementary Materials for:

    Wdpcp promotes epicardial EMT and epicardium-derived cell migration to facilitate coronary artery remodeling

    Xiangyang Liu, Ye Wang, Feng Liu, Min Zhang, Hejie Song, Bin Zhou, Cecilia W. Lo, Shilu Tong, Zhenlei Hu,* Zhen Zhang*

    *Corresponding author. Email: zhenzhang{at}sjtu.edu.cn (Z.Z.); 13564677103{at}163.com (Z.H.)

    This PDF file includes:

    • Fig. S1. Expression of Wdpcp in embryonic heart.
    • Fig. S2. Ciliary defects but normal transcriptional response to Shh signaling in Wdpcp mutant hearts.
    • Fig. S3. Normal formation of the intramyocardial endothelial plexus.
    • Fig. S4. Normal egression of coronary endothelial cells from the sinus venosus.
    • Fig. S5. Proliferative and apoptotic status of coronary endothelial cells in E13.5 hearts.
    • Fig. S6. Normal coronary vessel formation in E18.5 embryos with myocardium-specific deletion of Wdpcp.
    • Fig. S7. Wdpcp knockdown recapitulates the gel infiltration defect of Wdpcp mutant-derived EPDCs.
    • Table S1. List of qPCR primers used in the study.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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